Primary and secondary immunization with different physio-chemical forms of antigen

ABSTRACT

Animals, including humans, are immunized by antigens, for example, the HA antigen of influenza, by first administering to a naive animal a normally strongly-immunogenic form of the antigen, for example, inactiviated or attenuated whole cell virus and subsequently administering a normally weakly-immunogenic isolated and purified viral antigen, to achieve an enhanced immune response to the purified viral antigen.

This application is a continuation of application Ser. No. 07/943,247,filed Sep. 14, 1992 now abandoned.

FIELD OF INVENTION

The present invention relates to a novel immunization procedure foreliciting an immune response in animals, including humans.

BACKGROUND TO THE INVENTION

Vaccination is a procedure whereby an immune response to antigen can beachieved to protect a host from infection. Some antigens elicit a strongimmune response and some a weak response. Attempts have been made toenhance the immune response of weakly-immunogenic materials. The use ofchemical adjuvants achieves such potentiation but generally suchmaterials are toxic chemicals which cannot be used in humans.

Another procedure for achieving potentiation is to conjugate theweakly-immunogenic material to a strongly-immunogenic material andadminister the conjugate in a vaccine. For example, a conjugate of thecapsular polysaccharide of Haemophilus influenzae type b to diphtheriatoxoid, as described in U.S. Pat. Nos. 4,496,538 and 4,619,828, or aconjugate of a weak antigen to a monoclonal antibody targetingantigen-presenting cells, as described in U.S. Pat. No. 4,950,480, maybe employed.

SUMMARY OF INVENTION

In accordance with the present invention, there is provided a novelprocedure of vaccination to elicit an enhanced immune response to anormally weakly-immunogenic form of an antigen in an animal, byadministering the antigen to an animal primed with a highly-immunogenicform of the antigen.

The immunogenic form of the antigen is administered first to the naiveanimal to achieve a primary immune response to the antigen. Theweakly-immunogenic form of the antigen, generally an isolated purifiedantigen provided from natural sources or synthetically, which does notprovide a primary immune response in a naive animal, then isadministered to the primed animal to achieve a booster immune responseto the antigen. The term "weakly-immunogenic" as used herein refers bothto no immune response and a low immune response.

The present invention is applicable to a wide range of antigens whosedifferent physio-chemical forms produce different immune responses. Suchantigens may comprise viral, bacterial, fungal, protozan and parasiticproteins.

BRIEF DESCRIPTION OF DRAWINGS

FIGS. 1 and 2 contain graphical data of HAI titers achieved by variousforms of HA antigen in primed guinea pigs, as detail in Example 1 below;and

FIG. 3 contains graphical data of the immune response of various formsof OspA in mice.

GENERAL DESCRIPTION OF INVENTION

There exists isolated purified antigens which exhibit the antigenicityof the whole virus or other highly-immunogenic physio-chemical forms ofthe antigen but are either non-immunogenic or poorly immunogenic, i.e.they elicit no or only a small immune response, in naive animals,including humans, whereas the whole virus or other highly-immunogenicphysio-chemical forms of the antigen elicits a strong immune response.An example of such an antigen is the haemagglutinin antigen (HA) frominfluenza virus. The purified form of the HA is bromelain-cleaved toremove the hydrophobic tail HA(p).

Since the antigen is in pure form, it has little or no tendency toproduce any adverse side effects and hence is a desirable material touse for vaccination. The present invention enables such materials to beemployed in the vaccination of animals, including humans, againstdisease, by effecting a secondary immunization of the animal using thepurified antigen. The primary immunization of the animal is effectedusing a strong-immunogenic form of the antigen, usually the inactivatedor attenuated whole cell virus in the case of viral antigens.

The isolated purified form of the antigen may be prepared by separationfrom the whole cell or may be prepared synthetically, such as bychemical synthesis or by recombinant techniques. Among theweakly-immunogenic viral antigens which may be used in the invention arethe haemagglutinin antigen of influenza, and the gp120 protein ofretroviruses, especially HIV; as well as other viral proteins isolatedfrom viral membranes. Among the weakly-immunogenic bacterial antigenswhich may be used in the invention is the non-lipidated form of theouter surface protein A (OspA) of B. burgdorferi.

The invention is illustrated hereinafter with respect to thehaemagglutinin antigen (HA) from influenza virus as the booster antigenbut it will be apparent from the results given both for animal tests forthe HA antigen that the invention has application to a wide range ofantigens. Also presented below is data with respect to the immuneresponse to the outer surface protein A (OspA of B. burgdorferi.Lipidated OspA is a strong immunogen while non-lipidated OspA is not.However, an immune response to the OspA-NL was observed whenadministered as a booster to animals primed with OspA-L. The resultspresented show the generality of the procedure.

EXAMPLES

Example 1

This Example illustrates the effect of administration of HA to animals.

Guinea pigs were primed with either 1.0 μg of whole inactivated virus(results depicted in FIG. 1) or 1.0 μg of split HA i.e., FLUZONE®.(Split HA is provided by Triton X-100 detergent extraction of antigenfrom inactivated virus). The whole inactivated virus comprised an equalparts mixture of A/Taiwan and B/Panama formaldehyde inactivatedinfluenza virus particles. The doses of whole inactivated virus refer tothe dose of A/Taiwan material; (results depicted in FIG. 2). Three weekslater, the guinea pigs were given secondary immunization of eithersingle flu antigen or coadministered flu antigens. The results shown inFIGS. 1 and 2 indicate that co-administration does not enhance anti-HAresults in primed animals and hence the co-administration techniquedescribed in copending U.S. patent application Ser. No. 943,173 filedSep. 14, 1992 by Becker et al and assigned to the assignee hereof, isuseful only in naive animals if an enhanced immune response is to beachieved.

However, these results also show that the superior antigen for recallingmemory responses was HA(p) alone, while immunization with HA(p) at theprimary and secondary immunization did not generate a significant immuneresponse. These results show that HA(p) can recall memory immuneresponses to the HA antigen but cannot itself generate memory. HA(p) isnot immunogenic in naive animals or infants, even though it is antigenicin antibody-antigen reactions.

Example 2

The immunization procedure of Example 1 wherein various forms of HA orinfluenza infection were first administered to an animal followed by abooster administration was repeated in Swiss Webster mice. The resultsobtained are set forth in the following Table I:

                                      TABLE I                                     __________________________________________________________________________    Immunization of Normal and Previously Infected Mice with Flu Antigens         Primary                                                                              Secondary                                                                            Dose                                                                              HAI Titer.sup.(2)                                                                   HAI Titer                                                                          SN Titer                                                                           SN Titer.sup.(3)                            Immunization                                                                         Immunization                                                                         (ng)                                                                              3 wk  5 wk.sup.(4)                                                                       3 wk.sup.(4)                                                                       5 wk                                        __________________________________________________________________________    A/Taiwan                                                                             Fluzone ®                                                                        5   80    320  640  640                                         Infection                                                                     A/Taiwan                                                                             Fluzone ®                                                                        50  40    640  80   2560                                        Infection                                                                     A/Taiwan                                                                             HA(p)  450 80    1280 640  5120                                        Infection                                                                     HA(p)  HA(p)  450 5     5    5    5                                           Fluzone ®.sup.(1)                                                                Fluzone ®                                                                        5   5     10   5    20                                          Fluzone ®.sup.(1)                                                                Fluzone ®                                                                        50  10    40   5    80                                          __________________________________________________________________________     .sup.(1) FLUZONE ® is a registered trademark of Connaught                 Laboratories, Inc. for a split HA influenza vaccine prepared from the         A/Taiwan strain of influenza virus.                                           .sup.(2) HAI = haemagglutination inactivation                                 .sup.(3) SN = viral neutralization                                            .sup.(4) 3 week sera = primary immune response; 5 week sera = secondary       immune response                                                          

These results also show the booster immune response to HA(p) in micefirst immunized by whole inactivated virus.

Example 3

This Example illustrated the effect of administration of OspA inanimals.

The lipidated form of the OspA (OspA-L) protein of B. burgdorferi is avery potent immunogen in naive animals. Removal of the lipidated moietyfrom OspA (OspA-NL) dramatically decreases its immunogenicity in naiveanimals but not its antigenicity. OspA-L and OspA-NL were administeredin varying amounts to C3H/He mice (Taconic Laboratories) in primary andsecondary immunizations effected at days 0 and 21. The mice were bled atday 28. The dilution curves of an ELISA array of sera from the mice wereplotted graphically and the results are seen in FIG. 3. It is apparentfrom the data that the booster administration of OspA-NL produced animmune response in mice given a primary administration of OspA-L.

SUMMARY OF DISCLOSURE

In summary of this disclosure, the present invention provides a novelimmunization procedure which enables a strong immune response to beachieved from normally weakly-immunogenic purified viral proteins.

What we claim is:
 1. A method of achieving an immune reaction to ahighly-immunogenic form of a viral bacterial antigen in an animal, whichconsists essentially of the steps in the sequence setforth:administering by vaccination to said animal the highly-immunogenicform of said antigen to achieve a primary immune reaction to theantigen, wherein the highly-immunogenic form of said antigen isparticulate, and subsequently administering by vaccination to saidanimal a form of said antigen which is weakly-immunogenic and capable ofrecalling the memory immune reaction to the highly-immunogenic form ofthe antigen, to achieve a booster immune reaction to the antigen,wherein said weakly-immunogenic form of said antigen is soluble.
 2. Themethod of claim 1 wherein said highly immunogenic form of the antigen isinactivated or attenuated whole cell virus.
 3. The method of claim 2wherein said weakly-immunogenic form of the antigen is an isolated andpurified antigen.
 4. The method of claim 3 wherein said antigen isisolated and purified from whole cells.
 5. The method of claim 3 whereinsaid antigen is produced synthetically.
 6. The method of claim 3 wheresaid antigen is the HA(p) antigen from influenza virus.
 7. The method ofclaim 3 wherein said antigen is the gp120 protein from HIV.
 8. A methodof claim 1 wherein the antigen is a bacterial antigen.
 9. The method ofclaim 8 wherein said antigen is OspA-NL from B. burgdorferi.
 10. Themethod of claim 1 wherein said animal is a human.